The human immunodeficiency virus type-1 (HIV-1) is a complex retrovirus that encodes six regulatory proteins, including Tat, essential for viral replication. The Tat protein is a potent transcriptional activator of the HIV-1 long terminal repeat promoter element. A regulatory element between +1 and +60 in the HIV-1 LTR which is capable of forming a stable stem-loop structure designated TAR is critical for Tat function. In the absence of Tat, RNA polymerase II (pol II) terminates transcription prematurely. Tat-TAR interactions convert pol II into its processive form and lead to the efficient production of full-length viral transcripts. In addition to RNA-protein interactions, the regulatory function of Tat also requires protein-protein interactions between Tat and other cellular proteins. Therefore, interfering with the formation of functional RNA-protein and protein-protein complexes would inhibit the HIV gene expression. The goal of the proposed research is the discovery of low molecular weight compounds that inhibit the replication of HIV by binding to specific RNA and protein structures. The success of this research would provide opportunities to discover drugs for diseases such as cancer and AIDS. This project has three specific aims, which are as follows. Specific aim 1: Synthesis of encoded combinatorial libraries. Experiments are proposed to synthesize combinatorial chemical libraries using the one-bead-one-molecule method. Natural and unnatural amino acid residues will be incorporated during combinatorial ligand synthesis. A binary encoding system will be employed to determine the structure of active molecules. Specific Aim 2: Screening for RNA and protein ligands. Experiments are proposed to develop high throughput screening methods for identification of ligands that can target specific RNA and protein structures. Specific Aim 3: In vitro and in vivo activities of RNA and protein ligands. Fluorescence energy transfer methods will be developed to quantitatively determine the effects of small molecules in inhibition of functional RNA-protein and protein-protein complex formation. Inhibition of Tat trans-activation by small molecules will be determined in vitro in a cell-free transcription system and in vivo activities of small molecules will be assessed by monitoring the expression of marker genes in HeLa cell lines. The properties of lead compounds can be optimized and improved by analyzing the structure-function relationships governing inhibition of Tat trans-activation and target selectivity.